ObjectiveTo establish a non-invasive method based on fluorescent tracer technique using inducible co-stimulatory molecules(ICOS) expressed on activated T cells for diagnosing acute heart graft rejection in mice. MethodsThe cervical heterotopic heart transplantation was used as model to establish isograft, allograft, allograft plus tacrolimus treatment, and allograft with tacrolimus ceased groups. On the 1st, 3rd, 5th and 7th day after transplantation, Cy7.SE-ICOS-Ab was injected into the heart transplant mice via tail veins. The real-time fluorescent imaging changes of the graft were observed by fluorescent equipment. Flow cytometry was used to examine the expression of ICOS on spleen T cells of mice in each group. H-E staining was used to observe the pathological changes of cardiac graft. ResultsThere was no noticeable fluorescent imaging in the grafts at the 1st, 3rd, 5th and 7th day after transplantation in the isograft and allograft with tacrolimus treatment group. On the first day after transplantation, the fluorescent imaging of graft in the allograft group had no noticeable changes, but the fluorescent imaging gradually increased on the 3rd, 5th, and 7th day. The graft fluorescent imaging became stronger on the 3rd day after ceasing tacrolimus in the treated allograft group, and it became stronger at 5 and 7 days after ceasing tacrolimus. H-E staining found no noticeable rejection in isograft group and allograft plus tacrolimus treatment group at all time points. The allograft and allograft puls tacrolimus ceased group developed rejection on the 3rd day after transplantation, and the rejection became more serious on the 5th and 7th day. Flow cytometry showed that there were no significant differences in ICOS expression on spleen T cells on the 1st day after transplantation among the four groups (P>0.05). The isograft and allograft plus tacrolimus treatment group had no ICOS expression on the T cells, and ICOS expression in the allograft and allograft with tacrolimus ceased group gradually increased on the 3rd, 5th and 7th day. ConclusionICOS expression intensity is associated with the degree of graft rejection. Fluorescently labeled anti-ICOS can help to assess the development and severity of acute rejection after transplantation in a non-invasive way.